CAF1-5 Antibody

What is the CAF-1 complex and why is it important in chromatin research?

The Chromatin Assembly Factor-1 (CAF-1) is a heterotrimeric complex that plays a crucial role in nucleosome assembly during DNA replication and repair. It consists of three subunits: p150, p60, and p55. CAF-1 mediates chromatin assembly by depositing newly synthesized and acetylated histones H3 and H4 into nascent chromatin during DNA replication . This process is vital for maintaining genomic integrity and regulating gene expression, as proper nucleosome assembly directly affects DNA accessibility to transcription factors and other regulatory proteins . CAF-1 is also involved in heterochromatin maintenance in proliferating cells and plays a role in the coordinated inheritance of gene expression states . The study of CAF-1 provides critical insights into epigenetic regulation, DNA damage response, and cell cycle progression mechanisms.

Which CAF-1 subunit antibodies are available for research applications?

Several antibodies targeting different CAF-1 subunits are available:

• CAF-1 p150 antibodies: Available as recombinant monoclonal antibodies with various conjugates including Alexa Fluor® 488 . These target the largest subunit (p150/CHAF1A) which serves as the core component of the CAF-1 complex .

• CAF-1 p60 antibodies: Available as mouse monoclonal antibodies (such as B-10, clone designation) in multiple formats including unconjugated, agarose-conjugated, HRP-conjugated, and fluorophore-conjugated versions (FITC, PE, various Alexa Fluor® conjugates) .

• CAF-1 p55 antibodies: Available as polyclonal antibodies with various conjugates including biotin . These recognize the smallest subunit of CAF-1.
  Each antibody offers specific advantages depending on the experimental design and target species (human, mouse, rat) .

What experimental applications are supported by CAF-1 antibodies?

CAF-1 antibodies support a diverse range of experimental applications:

How should I optimize immunoprecipitation protocols to study CAF-1-interacting proteins?

For optimal immunoprecipitation of CAF-1 and its interacting proteins:

• Extract preparation: Prepare nuclear extracts from cells expressing CAF-1 (native or tagged versions). For cross-linking immunoprecipitation, harvest approximately 2×10^7 cells and cross-link with dithiobis(succinimidyl propionate) (2 mM) for 15 minutes on ice, followed by quenching with 50 mM Tris (pH 7.5) .

• Antibody selection: Use well-validated antibodies such as SS24 anti-p60 antibodies for native CAF-1, or anti-HA antibodies (e.g., 12CA5 or 3F10) for HA-tagged CAF-1 proteins .

• Immunoprecipitation conditions: Incubate extracts with antibody-conjugated beads for 3 hours at 4°C with rotation, followed by washing in appropriate buffers (e.g., Buffer A 100 for nuclear extracts or RIPA buffer for whole cell extracts) .

• Interactome analysis: Analyze co-precipitated proteins using mass spectrometry (LC-MS/MS) after either direct digestion of precipitates or fractionation using methods such as strong cation exchange chromatography .

• Validation of interactions: Validate mass spectrometry hits through in vitro co-immunoprecipitation using radiolabeled in vitro translation products to confirm direct interactions .

How can I distinguish between replication-coupled and repair-associated CAF-1 activity in my experiments?

Distinguishing between these two functions requires careful experimental design:

• Cell cycle synchronization: Synchronize cells at specific cell cycle phases using methods such as double thymidine block or serum starvation followed by release. CAF-1 activity during S-phase is predominantly replication-coupled .

• DNA damage induction: Induce DNA damage using UV irradiation, radiomimetic drugs, or site-specific endonucleases in non-S phase cells to specifically observe repair-associated CAF-1 recruitment.

• Co-localization analysis: Use dual immunofluorescence to analyze co-localization of CAF-1 (using antibodies such as Alexa Fluor® 488 Anti-p150) with:

  • PCNA for replication foci

  • γH2AX, 53BP1, or other DNA damage markers for repair sites

• PCNA-binding mutants: Utilize CAF-1 mutants defective in PCNA binding to distinguish between replication and repair functions, as PCNA interaction is crucial for both processes but can be differentially regulated.

• Quantitative analysis: Measure the kinetics of CAF-1 recruitment to chromatin after replication inhibition versus DNA damage induction to identify distinct temporal patterns.

What are common causes of non-specific binding with CAF-1 antibodies and how can they be minimized?

Non-specific binding with CAF-1 antibodies can arise from several sources:

• Antibody concentration: Excessive antibody concentrations often lead to increased background. Titrate antibodies carefully for each application – starting ranges typically include:

  • WB: 1:1000-1:5000 dilution (for concentrated antibodies like SS48 and SS53)

  • IF: 1:100-1:500 dilution

  • IP: 2-5 μg per sample

• Blocking optimization: Insufficient blocking is a common cause of non-specific binding. Optimize blocking using:

  • 5% non-fat dry milk or BSA in TBST for Western blots

  • 10% serum (from species different from antibody source) for immunofluorescence

  • 1% BSA for flow cytometry applications

• Antibody specificity: Confirm antibody specificity through:

  • Testing on CAF-1 knockout/knockdown samples as negative controls

  • Using alternative antibody clones targeting different epitopes

  • Pre-adsorption with immunizing peptides when available

• Cross-reactivity assessment: CAF-1 p55 shares structural similarities with other WD40 repeat proteins, potentially causing cross-reactivity. Validate signals using orthogonal methods or multiple antibodies targeting different subunits .

How should changes in CAF-1 localization patterns be interpreted following experimental manipulations?

Interpreting CAF-1 localization changes requires understanding its functional contexts:

• Diffuse nuclear to punctate pattern: Transition from diffuse nuclear staining to distinct nuclear foci typically indicates active recruitment to replication sites during S-phase or to DNA damage sites . This pattern change often correlates with increased chromatin assembly activity.

• Co-localization with heterochromatin: Enhanced association with heterochromatic regions (often co-staining with HP1 proteins) suggests a role in heterochromatin maintenance or assembly, particularly in late S-phase when heterochromatin replicates .

• Nucleolar exclusion/enrichment: Changes in nucleolar localization may indicate involvement in ribosomal DNA maintenance or repair processes.

• Cytoplasmic retention: Unexpected cytoplasmic localization might indicate disruption of nuclear import mechanisms or possible novel cytoplasmic functions.

• Quantitative assessment: For rigorous interpretation, quantify:

  • Percentage of cells showing specific patterns

  • Signal intensity at specific cellular compartments

  • Co-localization coefficients with markers of interest

  • Temporal dynamics using live-cell imaging when possible

What controls are essential when using CAF-1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Robust ChIP experiments with CAF-1 antibodies require comprehensive controls:

• Antibody validation controls:

  • Input sample (typically 1-10% of starting material)

  • No-antibody (beads-only) control to assess non-specific binding

  • Isotype control antibody (matching the CAF-1 antibody class and species)

  • ChIP using cells depleted of CAF-1 (siRNA/shRNA treated)

• Locus-specific controls:

  • Actively replicating regions (positive control for replication-coupled CAF-1)

  • Transcriptionally inactive regions (potential enrichment for heterochromatin-associated CAF-1)

  • Constitutively active housekeeping genes (typically negative control)

• Experimental validation:

  • Sequential ChIP (re-ChIP) to confirm co-occupancy with known partners (PCNA, newly synthesized histones)

  • Complementary techniques like DNA combing or FAIRE to correlate with replication timing

• Data analysis recommendations:

  • Normalize to input and IgG control

  • Present data as fold enrichment over background

  • Perform biological replicates (minimum three) for statistical validity

  • Consider cell cycle phase when interpreting results, as CAF-1 chromatin association is highly cell cycle-dependent

How are recent advances in microscopy enhancing our understanding of CAF-1 dynamics?

Advanced microscopy techniques are revolutionizing CAF-1 research:

• Super-resolution microscopy: Techniques like STORM, PALM, and STED overcome diffraction limits, revealing previously undetectable CAF-1 substructures at replication foci. These methods require highly specific fluorophore-conjugated antibodies, such as the Alexa Fluor® 488 Anti-p150 CAF1/CAF antibody .

• Live-cell imaging: CRISPR-mediated endogenous tagging of CAF-1 subunits with fluorescent proteins enables real-time tracking of CAF-1 dynamics without antibody-based detection limitations.

• Single-molecule tracking: Techniques using photoactivatable fluorophore-conjugated antibodies allow tracking of individual CAF-1 complexes, revealing diffusion rates, residence times, and binding kinetics at specific chromatin regions.

• FRAP and FLIP analyses: These techniques measure CAF-1 mobility and exchange rates between chromatin-bound and soluble pools, providing insights into functional dynamics during replication and repair.

• Correlative light-electron microscopy: This approach combines fluorescence imaging of antibody-labeled CAF-1 with ultrastructural analysis, revealing its precise localization relative to chromatin ultrastructure. Future methodological improvements should focus on developing antibodies with even greater specificity and sensitivity, particularly for studying the less-characterized p55 subunit, and expanding the range of available conjugates for multiplexed imaging applications.